Pain conditions are a major health problem in the US and lead to medical morbidity and a reduced quality of life for millions of Americans. Chronic neuropathic pain conditions are especially difficult to treat. A largely unaddressed challenge is how the transition from acute pain to chronic neuropathic pain occurs and how to prevent and reverse this transition in patients. Spinal cord synaptic plasticity and long-term potentiation (LTP) have been strongly implicated in chronic neuropathic pain development. Accumulating evidence also points to an important role of glial cells in the pathogenesis of neuropathic pain. Astrocytes are the most abundant cell type in the CNS and maintain the homeostasis of the CNS. It is well established that astrocytic hemichannels such as connexin-43 (Cx43) constitute an important pathway for gliotransmitter release. Although Cx43 was typically thought to regulate gap junction communication between astrocytes, this function could be switched to paracrine signaling via ATP and glutamate release under injury conditions. Our central hypothesis is hemichannels-mediated gliotransmitter release after nerve injury contributes to transition from acute pain to chronic neuropathic pain by modulating spinal cord synaptic plasticity and LTP. We will test our central hypothesis by addressing the following 4 specific aims: Aim 1 will test the hypothesis that spinal nerve injury increases glutamate and ATP release from spinal cord astrocytes; Aim 2 will test the hypothesis that Cx43-mediated astrocytic ATP release plays a chief role in microglia activation and microgliosis in the spinal cord after spinal nerve injury; Aim 3 will determine the role of Cx43-medicated astrocytic gliotransmitter release in spinal cord synaptic plasticity and LTP after nerve injury; Aim 4 will define the role o astrocytic Cx43 in neuropathic pain development and maintenance after nerve injury. This proposal will involve formation of an innovative partnership between Dr. Ji, a pain scientist with expertise in studying neuronal-glial interactions and neural plasticity in neuropathic pain, and Dr Nedergaard with expertise in studying astrocytic ATP and glutamate release after spinal cord injury and stroke. This application will employ a multidisciplinary approach including the use of inducible transgenic mice with genetically modified astrocytes, in vivo imaging of ATP release (bioluminescence) and microglia motility and Ca2+ changes (2-photon) in the spinal cord, behavioral testing of evoked and ongoing neuropathic pain after nerve injury, and ex vivo and in vivo electrophysiology in the spinal cord. The proposed studies will provide a step-by-step analysis of neuron- glia interactions initiated by nerve injury and may comprise an efficient means to prevent and treat chronic pain.